Microorganisms isolated from patients and other sources are routinely tested for their susceptibility to antimicrobial agents and for their metabolic growth requirements. In particular, the minimum inhibitory concentrations (MIC's) of antimicrobial agents or the categorical interpretations (susceptible, moderately susceptible, or resistant) of microorganisms isolated from clinical sources are routinely determined.
Numerous methods and apparatus have been developed to conduct susceptibility tests. In particular, multi-compartmentalized devices, such as microdilution panels, with each compartment containing a specific quantity of an antimicrobial agent or a growth promoting material, such as a vitamin, are often used to determine growth and susceptibilities. These compartments generally contain, in addition to the agent investigated, growth supporting medium. These devices may be freshly prepared, frozen or dried for convenient storage.
To conduct susceptibility tests the above described devices are inoculated with a standardized microbiological inoculum and incubated until visible growth appears. This typically takes 15-24 hours. Generally, microbial growth in either a liquid medium or on the surface of a solid support medium is determined by direct visual recognition or by turbidometric measurements. The endpoint of susceptibility tests is defined as the lowest concentration of an antimicrobial agent in which growth, when compared to that of the growth compartment, appears to be inhibited.
Commonly used susceptibility tests require 15-24 hours of incubation prior to the availability of results. Earlier receipt by the physician of accurate antimicrobial agent susceptibility information would result in better patient treatment. A number of methods have been published to obtain earlier susceptibility determinations. They may be divided into measurement of bacterial mass described in U.S. Pat. Nos. 4,236,211 (Pfizer), 3,832,532 (Pfizer), G.B. Patent No. 1,554,134 (Pfizer), and indirect estimation of bacterial mass by measurement of enzymatic activity described in U.S. Pat. No. 4,242,447 (Bio Research), fluorogenic measurement of phosphate, U.S. Pat. No. 3,509,026 (Litton Systems Inc.), fluorogenic measurement of phosphotase, FR 2,504,679, use of methylumbelliferyl derivatives, EP 0,091,837B and in articles describing the use of amido-coumarin derivatives for antibiotic susceptibility testing by M. R. Mateo, et al., Abstract Annual Meeting, American Society for Microbiology, 1980, C201, P. 308 and the measurement of metabolic activity by reduction of tetrazolium in microdilution panels by T. Urban and C. Jarstrand, J. Antimicro. Chemo (1981) 8, 363-369. All the following are about the use of a mixture of fluorogenic substrates: Nolte et al., J. Clin. Microbio. (1988) 26, 1079-1084, Staneck et al., J. Clin. Microbiol. (1985), 187-191, Doern et al., J. Clin. Micro. (1987), 1481-1485, and Staneck et al., J. Clin. Micro. (1988) v. 26 1-7.
One particular method is the Sensititre.TM. system which has an instrument capable of automatically reading antimicrobial susceptibility microdilution trays. In this procedure, microbial growth is determined by the measurement of fluorescence produced by bacterial enzymatic action on fluorogenic substrates. The fluorescence signals are interpreted by the instrument and converted to MIC's. Staneck, (1985) Supra at 187. The Sensititre.TM. method involves the use of a fluorogenic substrate cocktail to detect the minimal inhibitory concentration for Gram positive and Gram negative bacteria based on a single measurement within five hours of the addition of the hydrolysable fluorogenic substrates to the inoculum. It is disclosed that the fluorogenic substrates for this group of bacteria are selected from 7-(N)-(aminoacyl)-7-amido-4-methylcoumarin, 4-methylumbelliferyl nonanoate, 4-methylumbelliferyl phosphate. EP 0,091,837B.
Single measurement methods to predict or determine minimal inhibitory concentration using fluorogenic substrates, however, have been found to be unreliable. In order to determine accurate minimal inhibitory concentrations sufficient growth and utilization of the substrate must occur. A single measurement precludes the accurate determination of a minimal inhibitory concentration under optimum conditions because different bacterial species may obtain sufficient growth to determine minimal inhibitory concentrations at different times. A single early measurement may result in an inaccurate prediction of a minimal inhibitory concentration because of insufficient expression of resistance. A single late measurement may result in an inaccurate estimation of enzymatic activity in the antimicrobial agent containing compartment in relation to that of the growth compartment. After a certain fluorescence level is reached, the photometric detection system is unable to accurately determine fluorescence. Consequently, a need exists to develop a system to accurately predict minimal inhibitory concentrations for a wide range of Gram positive organisms using fluorogenic substrates in one standardized test system.